Device for immunochromatographic analysis

ABSTRACT

Methods are disclosed for conducting assays. One such method comprises providing in combination a first bibulous member zone (&#34;first zone&#34;) and a liquid medium containing a component. The first zone has non-diffusively bound thereto a reagent interreactive with the component. Conditions are selected wherein the liquid medium and at least a portion of the component contained therein traverse all of the first zone and migrate by capillary migration into a second bibulous member zone (&#34;second zone&#34;). The second zone is of a different composition than the first zone and is incapable of specifically binding the component except when an analyte is to be detected and the method further includes causing a reagent to become bound to the first bibulous member zone in relation to the amount of analyte present. The distance the component has migrated into the second zone or the difference in the distances the medium and the component have migrated into the second zone is determined, the distance or the difference being related to the amount of the component in the liquid medium or the amount of the reagent.

CROSS-REFERENCE TO RELATED APPLICATION

This is a Divisional appplication of U.S. patent application Ser. No.241,307 filed May 10, 1994, now U.S. Pat. No. 5,451,507, which is aContinuation application of U.S. patent application Ser. No. 940,137,filed Sep. 3, 1992, now U.S. Pat. No. 5,334,513, which is a Divisionalof application Ser. No. 07/376,723, filed Jul. 7, 1989, now U.S. Pat.No. 5,164,294, which is a Continuation-in-Part of application Ser. No.07/194,708, filed May 17, 1988, now U.S. Pat. No. 5,039,607, thedisclosures of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The ability to employ naturally occurring receptors or antibodiesdirected to specific compounds in assaying for the presence of acompound of interest has created a burgeoning immunoassay business. Ineach of the assays, a complementary pair of specific binding pair("sbp") members, usually an immunological pair, involving a ligand and areceptor (antiligand) is involved, wherein one of the sbp members islabeled with a label which provides a detectable signal. The immunoassaymethodology results in a distribution of the signal label between signallabel bound in a complex of the sbp members and unbound signal label.The differentiation between bound and unbound signal label can be as aresult of physical separation of bound from unbound signal label ormodulation of the detectible signal between bound and unbound signallabel.

For the most part, immunoassays have been directed to quantitativedetermination of a wide variety of compounds of interest in clinicallaboratories requiring relatively sophisticated equipment and carefultechnique. Immunoassays have found less extensive commercial applicationwhere semi-quantitative or qualitative results would be acceptable andthe determination would involve non-laboratory personnel, such as in ahome or a medical practitioner's office. Even in the clinicallaboratory, simple and rapid screening tests employing inexperiencedpersonnel could serve to provide substantial economies.

In developing an immunoassay, there are many considerations. Oneconsideration is to provide substantial differentiation between theobserved signal resulting from signal label when bound as compared tounbound. Another consideration is to minimize interference fromendogenous materials in the sample suspected of containing the compoundof interest. A further consideration is the ease with which the observedsignal can be detected and serve to differentiate between concentrationsin the concentration range of interest. Another consideration is theconcentration of the compound of interest in a sample. Other factorsinclude the ease of preparation of the reagents, the precision withwhich samples and reagent solutions must be prepared and measured, thestorage stability of the reagents, the number of steps required in theprotocol, and the proficiency and accuracy with which each of the stepsmust be performed. Therefore, in developing an assay that can haveapplication with untrained personnel, such as assays to be performed inthe home, in forensic medicine, by medical practitioners, or the like,the observed result should be minimally affected by variations in themanner in which the protocol is carried out and the techniques forperforming the various steps should be simple.

In general, immunoassays that permit the determination of a diluteanalyte in a sample have been difficult to design and those that havebeen demonstrated require numerous steps and long periods of time.

Frontal analysis (or breakthrough analysis) is a known method forquantitating active groups on a solid support in a chromatography column(C. R. Lowe and P. D. G. Dean, Affinity Chromatography, John Wiley &Sons, Ltd., New York, 1974). In frontal analysis, a solution of thereacting species at a known concentration is applied to the column.Measurement of the volume needed to saturate the column so that thereacting species is detected in the column effluent (the breakthroughvolume) allows determination of the active group concentration on thecolumn.

2. Description of the Related Art

U.S. Pat. No. 4,425,438 describes a method and device for assaying atest substance utilizing a primary absorbent substance for selectivelyallowing only a quantity of an analytical reagent proportional to thequantity of test substance to pass therethrough when test substance andanalytical reagent are contacted with the primary absorbent. Ananalytical absorbent is disposed in a series of zones for sequentiallyabsorbing the analytical reagent which passes through the primaryabsorbent so that detection of the last zone of absorbed analyticalreagent indicates the quantity of test substance. In the method testsubstance and analytical reagent are passed through the primaryabsorbent and then the analytical absorbent followed by detection of thelast zone in which analytical reagent is absorbed. The device is afunnel with the primary absorbent therein for directing the testsubstance and analytical reagent to a narrow tube holding the analyticalabsorbent.

A test device for determining a characteristic of a sample, particularlyfor determining substances in fluid samples, is disclosed in U.S. Pat.No. 4,094,647. A thin layer chromatography device and method of making achromatography test is disclosed in U.S. Pat. No. 4,384,958. Animmunoassay wherein labeled antibody is displaced from immobilizedanalyte analog is described in U.S. Pat. No. 4,434,236. A device andmethod for detecting myoglobin is disclosed in U.S. Pat. No. 4,189,304.Test strips for analyzing substances dissolved in liquids are describedin U.S. Pat. No. 4,438,067. A multi-layered test device for determiningthe presence of a liquid sample component and the method of using such adevice, are described in U.S. Pat. No. 4,160,008. A method for measuringantigen by labeled antigen using insoluble antibody is disclosed inJapanese Patent Application Laid-Open No. 5925/73--Jan. 25, 1973.

A concentrating zone method in heterogeneous immunoassays is disclosedin U.S. Pat. No. 4,366,241. U.S. Pat. No. 4,168,146 describes animmunoassay test strip. U.S. Pat. Nos. 3,990,850 and 4,055,394 describediagnostic test cards. An automated method for quantitative analysis ofbiological fluids is described in U.S. Pat. No. 4,327,073. A chromogenicsupport immunoassay is disclosed in International Application No.PCT/US83/01887. A heterogeneous immunoassay for digoxin using ouabain asa separation means is described in U.S. Pat. No. 4,551,426.

A wide variety of patents and patent applications provide an extensiveliterature of different techniques for producing detectible signals inimmunoassays. The following list is merely illustrative of some of thesetechniques which can find application in this invention. The followingis a list of United States patents and patent applications and a generalstatement of the type of label involved:

U.S. Pat. Nos. 3,646,346, Radioactive Label; 3,654,090, 3,791,932 and3,817,838, Enzyme Labels; 3,996,345, Fluorescer-Quencher Labels;4,062,733, Radioactive Label; 4,067,959, Fluorescer or Enzyme Label;4,104,029, Chemiluminescent Label; and 4,160,645, Non-Enzymatic CatalystLabel. See U.S. Pat. Nos. 3,966,879 for an electrophoretic techniqueemploying an antibody zone and 4,120,945 for an RIA where labeledanalyte is initially bound to a solid support through antibody. U.S.Pat. No. 4,233,402 employs enzyme pair labels; U.S. Pat. No. 4,720,450,chemically induced fluorescent labels; and U.S. Pat. No. 4,287,300,enzyme anionic charge labels.

SUMMARY OF THE INVENTION

The methods of the present invention are useful in conducting assays.One such method for conducting an assay comprises providing incombination a first bibulous member zone ("first zone") and a liquidmedium containing a component. The first zone has non-diffusively boundthereto a reagent interreactive with the component. Conditions areutilized wherein the liquid medium and at least a portion of thecomponent contained therein traverse all of the first zone and migrateinto a second bibulous member zone ("second zone") by capillarymigration. The second zone is of different composition than that of thefirst zone and is incapable of specifically binding the component exceptwhen an analyte is to be detected and the method further includescausing a reagent to become bound to the first bibulous member zone inrelation to the amount of analyte present. The distance the componenthas migrated into the second zone or the difference in the distances themedium and the component have migrated into the second zone isdetermined. The distance or the difference is related to the amount ofthe component in the liquid medium or the amount of the reagent.

Another such method for determining the amount of a component in aliquid medium, comprises contacting a first bibulous member zone ("firstzone") with a liquid medium containing a component. The first zone hasnon-diffusively bound therein a reagent interreactive with thecomponent. The contact is carried out under conditions wherein theliquid medium and at least a portion of the component contained thereintraverse all of the first zone and migrate into a second bibulous memberzone ("second zone") by capillary migration. The second zone is ofdifferent composition than the first zone and is incapable ofspecifically binding the component except when an analyte is to bedetected and the method further includes causing a reagent to becomebound to the first bibulous member zone in relation to the amount ofanalyte present. The second zone may be chemically reactive with thecomponent. The distance the component has migrated into the second zoneor the difference in the distance the medium and the component havemigrated into the second zone is measured. The distance or thedifference is related to the amount of the component in the liquidmedium.

Another such method for conducting an assay comprises combining asolution of an analyte with a first bibulous member wherein a reagentcapable of reacting with the analyte becomes non-diffusively bound tothe first bibulous member. A liquid medium containing a component iscombined with the first bibulous member wherein the componentinterreacts with the reagent. The first bibulous member is in liquidreceiving relationship with, or is a spacially separated portion of, asecond bibulous member of different composition than that of the firstbibulous member and incapable of specifically binding the reagent.Conditions are chosen wherein the liquid medium and the component firsttraverse the first bibulous member and then at least a portion of thesecond bibulous member by capillary action. A border defined by theconcentration of the component, or a reaction product thereof, on thesecond bibulous member is determined. The position of the border isrelated to the amount of analyte in the solution or the amount ofreagent on the first bibulous member.

Another such method is useful in assaying for the presence of an analytein a sample suspected of containing the analyte. In one embodiment ofthe method of the present invention, a portion of a first bibulousmember is contacted with a first liquid, usually aqueous, medium orsolution containing a first reagent such as a specific binding pair(sbp) member to cause the first reagent to become bound to the firstbibulous member. The first bibulous member can be in, or be placed in,liquid receiving relationship with an absorbent member. If the firstbibulous member is in liquid receiving relationship with the secondbibulous member prior to contact of the first bibulous member with thefirst medium, then the first reagent must be capable of binding to theanalyte to form bound and unbound portions of the first reagent, whereinthe unbound portion is the first reagent in the first medium. The firstreagent is then caused to become bound to the first bibulous member. Thecontact is carried out under conditions wherein the first mediumtraverses or flows through the first bibulous member and at least aportion of the absorbent member by capillary action. Next, the firstbibulous member is brought into liquid receiving relationship with asecond bibulous member if it is not already in such a relationship. Aportion of the first bibulous member is contacted with the secondliquid, usually aqueous, medium under conditions wherein the secondaqueous medium traverses the first bibulous member by capillary actionand at least a portion of the second bibulous member. In this way acomponent in the second medium is absorbed by and preferably becomesnon-diffusely bound to the second bibulous member in relation to thepresence of analyte in the first medium. The presence of the componenton at least a portion of the second bibulous member is determined and isindicative of the presence of analyte. Furthermore, the distance thecomponent traverses the second bibulous member is related to the amountof analyte in the first medium.

In another aspect of the present invention a combination is provided ina first liquid, usually aqueous, medium or solution. The combinationincludes (1) at least a portion of a first bibulous member, at least aportion thereof having a first reagent, for example, an sbp member,non-diffusively bound thereto, and (2) a sample suspected of containingan analyte. When the analyte is not capable of binding to the firstreagent, a second reagent capable of binding to the analyte and to thefirst reagent is present in the medium or on the first bibulous member.The first bibulous member is in liquid receiving relationship with anabsorbent member. Conditions are chosen such that the first medium orsolution traverses or flows through the first bibulous member and atleast a portion of the absorbent member by capillary action. The firstbibulous member is then brought into liquid receiving relationship witha second bibulous member. Another combination is provided in a secondliquid, usually aqueous, medium. This combination includes at least aportion of the first bibulous member and a component that is capable ofbinding to the first or second reagent in relation to the presence ofthe analyte in the first medium. The second bibulous member preferablyhas a specific binding partner for the component non-diffusively boundto at least a portion thereof. The combination is provided underconditions wherein the second liquid medium traverses at least a portionof the first bibulous member and at least a portion of the secondbibulous member by capillary action. The distance the componenttraverses the second bibulous member is determined and is related to theamount of analyte in the sample.

In another aspect of the present invention, the liquid receivingrelationship between the first bibulous member and an absorbent memberis terminated prior to bringing the first bibulous member into contactwith a second bibulous member.

In another embodiment of the present invention, the second bibulousmember is a chromatographic member such as an immunochromatographicmember and a border is determined on the chromatographic member. Theposition of the border is related to the amount of analyte in the firstmedium or the amount of component in the liquid medium or the amount ofreagent on the first bibulous member.

Another aspect of the present invention concerns a method for conductingan assay for an analyte, wherein there is provided in combination in afirst liquid, usually aqueous, medium (1) a portion of a bibulous memberto at least a portion of which is non-diffusively bound a fist specificbinding pair (sbp) member and (2) a sample suspect of containing ananalyte. A first reagent may include a conjugate comprising a second sbpmember capable of binding the analyte and a third spb membercomplementary to the first sbp member, which is present in the medium oron the bibulous member. The bibulous member is in liquid receivingrelationship with an absorbent member. The first medium traverses thebibulous member and at least a portion of the absorbent member bycapillary action and the conjugate becomes non-diffusively bound to thebibulous member. The liquid receiving relationship between the bibulousmember and the absorbent member is terminated. Another combination isprovided in a second liquid, usually aqueous, medium, which includes aportion of the first bibulous member and a component. The componentcomprises an analyte analog bound to, or capable of becoming bound to, amember of a signal producing system. The bibulous member is in liquidreceiving relationship with an immunochromatographic member havingnon-diffusively bound thereto a binding partner for the component. Underthe conditions employed, the second medium traverses the bibulous memberand at least a portion of the immunochromatographic member by capillaryaction. The bibulous member is in liquid receiving relationship with theabsorbent member and the immunochromatographic member at substantiallydifferent times. A border is determined on the immunochromatographicmember, wherein the position of the border is related to the amount ofanalyte in the first medium.

Another aspect of the present invention concerns a method for conductingan assay for an analyte, wherein there is provided in combination in afirst liquid, usually aqueous, medium or solution (1) a portion of afirst bibulous member to which is non-diffusively bound a first reagentsuch as, for example, an analyte analog, and (2) a sample suspected ofcontaining an analyte. A second reagent capable of binding said analyteis present in the solution or on the bibulous member. The bibulousmember is in liquid receiving relationship with an absorbent member. Thefirst medium or solution traverses or flows through the bibulous memberand at least a portion of an absorbent member by capillary action andthe second reagent becomes non-diffusively bound to the bibulous memberin inverse proportion to the amount of the analyte in the first mediumor solution. The liquid receiving relationship between the bibulousmember and said absorbent member is terminated. Another combination isprovided in a second liquid, usually aqueous, medium, which includes aportion of said bibulous member and a component. The component includes,or is capable of having bound to it, a member of a signal producingsystem, and is capable of binding to the first or second reagent boundto the bibulous member. The bibulous member is in liquid receivingrelationship with an immunochromatographic member having non-diffusivelybound thereto a specific binding partner for the component. Under theconditions employed, the second medium traverses the bibulous member andat least a portion of the immunochromatographic member by capillaryaction. The bibulous member is in liquid receiving relationship with theabsorbent member and the immunochromatographic member at substantiallydifferent times. A border is determined on the immunochromatographicmember, wherein the position of the border is related to the amount ofanalyte in the first medium.

In another embodiment a combination is provided comprising (1) abibulous member to which is non-diffusively bound an antibody for ananalyte, and (2) a first liquid, usually aqueous, medium or solutionsuspected of containing an analyte. The bibulous member is in liquidreceiving relationship with an absorbent member. Conditions are chosento allow the first medium to traverse the bibulous member and at least aportion of the absorbent member by capillary action. The liquidreceiving relationship between said bibulous member and the absorbentmember is terminated. Another combination is provided in a secondliquid, usually aqueous, medium that includes a portion of the bibulousmember and a component. The component is bound to, or capable ofbecoming bound to, a member of a signal producing system and is capableof binding to the antibody for said analyte. The bibulous member is inliquid receiving relationship with an immunochromatographic memberhaving non-diffusively bound thereto a specific binding partner for thecomponent. Under the conditions of the method the second mediumtraverses the bibulous member and at least a portion of theimmunochromatographic member by capillary action, with the proviso thatthe bibulous member is in liquid receiving relationship with theabsorbent member and the immunochromatographic member at substantiallydifferent times. A border is determined on the immunochromatographicmember. The position of the border is related to the amount of analytein the first medium or solution, the amount of reagent, or the amount ofcomponent in the second medium.

Another aspect of the present invention concerns a method for conductingan assay for digoxin. The method comprises contacting an end portion ofa bibulous strip having a zone distal from the end portion, wherein thezone has an antibody (Ab₁) for an organic molecule of molecular weightless than 1500 non-diffusively bound thereto, with a first aqueousmedium or solution suspected of containing digoxin. A conjugatecomprising an antibody for digoxin conjugated or bound to the organicmolecule is present in the first medium or on the bibulous strip. Underthe conditions chosen the first medium or solution traverses bycapillary action the bibulous strip and at least a portion of anabsorbent member in liquid receiving relationship with the strip and theconjugate becomes non-diffusively bound to Ab₁, and digoxin, if present,becomes bound to the conjugate. The liquid receiving relationshipbetween the bibulous member and the absorbent member is terminated. Acombination is provided in a second liquid, usually aqueous, medium thatcomprises the end portion of the bibulous strip and a componentcomprising a digoxin analog bound to an enzyme. The bibulous strip is inliquid receiving relationship with an immunochromatographic strip havingnon-diffusively bound thereto an antibody for digoxin. The second mediumis allowed to traverse the bibulous strip and at least a portion of theimmunochromatographic member by capillary action, with the proviso thatthe strip is in liquid receiving relationship with the absorbent memberand the immunochromatographic strip at substantially different times.Again, a border on the immunochromatographic strip is determined whereinthe position of the border is related to the amount of analyte in saidsample.

In another embodiment of an assay for digoxin, an end portion of abibulous strip to which is non-diffusively bound a digoxin analog iscontacted with a first liquid, usually aqueous, medium or solutioncontaining a sample suspected of containing digoxin. An antibody fordigoxin (Ab_(D)) is present in the first medium or on the bibulousstrip. The bibulous strip is in liquid receiving relationship with anabsorbent member and conditions are chosen wherein the first mediumtraverses the bibulous member and at least a portion of the absorbentmember by capillary action and the antibody Ab_(D) becomesnon-diffusively bound to the digoxin analog when digoxin is not presentin the sample. The liquid receiving relationship between the bibulousstrip and the absorbent member is terminated. A combination is providedin a second liquid, usually aqueous, medium and includes the end portionof the bibulous strip and a conjugate comprising an antibody for Ab_(D)bound to an enzyme. The bibulous strip is in liquid receivingrelationship with an immunochromatographic strip having non-diffusivelybound thereto a receptor for the antibody for Ab_(D). Under theconditions of the assay, the second medium traverses the bibulous stripand at least a portion of the immunochromatographic strip by capillaryaction. The bibulous strip is in liquid receiving relationship with theabsorbent member and the immunochromatographic strip at substantiallydifferent times. A border is determined on the immunochromatographicstrip. The position of the border is related to the amount of analyte inthe first medium.

In another embodiment of a method in accordance with the invention, anend portion of a bibulous member to which is non-diffusively bound anantibody for digoxin is contacted with a first aqueous medium orsolution containing a sample suspected of containing digoxin. Thebibulous strip is in liquid receiving relationship with an absorbentmember and conditions are chosen such that the first medium traversesthe bibulous member and at least a portion of the absorbent member bycapillary action. The liquid receiving relationship between the bibulousmember and the absorbent member is terminated. In a second liquid,usually aqueous, medium a combination of the end portion of the bibulousstrip and a conjugate comprising a digoxin analog bound to an enzyme isformed. The bibulous strip is in liquid receiving relationship with animmunochromatographic strip having a receptor for conjugatenon-diffusively bound thereto. The second medium traverses the bibulousstrip and at least a portion of the immunochromatographic strip bycapillary action. The bibulous strip is in liquid receiving relationshipwith the absorbent member and the immunochromatographic strip atsubstantially different times. Again, a border on saidimmunochromatographic strip is determined, the position of which isrelated to the amount of analyte in said first medium.

The invention further includes a device for analyzing for the presenceor amount of an analyte, a reagent, or a component in a liquid medium.The device comprises a first bibulous member, an absorbent member, and asecond bibulous member. The bibulous member is capable of being inalternating liquid receiving relationship with the absorbent member andthe second bibulous member. Kits for carrying out the methods of thepresent invention are also included in the present invention.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a schematic of a device in accordance with the presentinvention.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

As mentioned above, the present invention is directed to methods,devices and kits for determining the presence or amount, at or above apredetermined minimum detectible amount, of a component in a liquidmedium, a reagent, and/or an analyte in a sample suspected of containingthe analyte. The present invention is particularly applicable to thedetermination of relatively dilute components in a medium, reagents on asurface or analytes in a sample. The present invention provides a meansfor avoiding sample interference and results in an enhanced sensitivityand a quicker method than those of the prior art. The present inventionalso provides means for greater balance between the number of analytemolecules in the sample and the number of molecules that will ultimatelyresult in signal formation.

Before proceeding further with the description of the specificembodiments of the present invention, a number of terms will be defined.

Analyte--a compound or composition to be measured that is capable ofbinding specifically to a binding member such as an antibody orchelating agent, usually an antigen or drug, or a composition that ischemically reactive such as a molecule or ion capable of oxidation orreduction.

The precise nature of the antigenic and drug analytes together withnumerous examples thereof are disclosed in U.S. Pat. No. 4,299,916 toLitman, et al., particularly columns 16 to 23, and in U.S. Pat. No.4,275,149, columns 17 and 18, the disclosures of which are incorporatedherein by reference.

The analytes include ligands and receptors that are characterized byhaving single binding sites (monovalent) or multiple binding sites(polyvalent). The polyvalent analytes will normally be poly(aminoacids), i.e., polypeptides and proteins, polysaccharides, nucleic acids,and combinations thereof. Such combinations or assemblages includebacteria, viruses, chromosomes, genes, mitochondria, nuclei, cellmembranes, and the like.

A wide variety of proteins may be considered as to the family ofproteins having similar structural features, proteins having particularbiological functions, proteins related to specific microorganisms,particularly disease causing microorganisms, etc.

The monoepitopic ligand analytes will generally be from about 100 to2,000 molecular weight, more usually from 125 to 1,000 molecular weight.The analytes of interest include drugs, metabolites, pesticides,pollutants, and the like. Included among drugs of interest are thealkaloids. Among the alkaloids are morphine alkaloids, which includesmorphine, codeine, heroin, dextromethorphan, their derivatives andmetabolites; cocaine alkaloids, which include cocaine and benzoylecogonine, their derivatives and metabolites, ergot alkaloids, whichinclude the diethylamide of lysergic acid; steroid alkaloids; iminazoylalkaloids; quinazoline alkaloids, isoquinoline alkaloids; quinolinealkaloids, which include quinine and quinidine; diterpene alkaloids,their derivatives and metabolites.

The next group of drugs includes steroids, which includes the estrogens,estrogens, androgens, andreocortical steroids, bile acids, cardiotonicglycosides and aglycones, which includes digoxin and digoxigenin,saponins and sapogenins, their derivatives and metabolites. Alsoincluded are the steroid mimetic substances, such as diethylstilbestrol.

The next group of drugs is lactams having from 5 to 6 annular or ringmembers, which include the barbituates, e.g. phenobarbital andsecobarbital, diphenylhydantonin, primidone, ethosuximide, and theirmetabolites.

The next group of drugs is aminoalkylbenzenes, with alkyl of from 2 to 3carbon atoms, which includes the amphetamines, catecholamines, whichincludes ephedrine, L-dopa, epinephrine, narceine, papaverine, and theirmetabolites.

The next group of drugs is benzheterocyclics which include oxazepam,chlorpromazine, tegretol, imipramine, their derivatives and metabolites,the heterocyclic rings being azepines, diazepines and phenothiazines.

The next group of drugs is purines, which includes theophylline,caffeine, their metabolites and derivatives.

The next group of drugs includes those derived from marijuana, whichincludes cannabinol and tetrahydrocannabinol.

The next group of drugs includes the vitamins such as A, B, e.g., B₁₂,C, D, E and K, folic acid, and thiamine.

The next group of drugs is prostaglandins, which differ by the degreeand sites of hydroxylation and unsaturation.

The next group of drugs is antibiotics, which include penicillin,chloromycetin, actinomycetin, tetracycline, terramycin, the metabolitesand derivatives.

The next group of drugs is the nucleosides and nucleotides, whichinclude ATP, NAD, FMN, adenosine, guanosine, thymidine, and cytidinewith their appropriate sugar and phosphate substituents.

The next group of drugs is miscellaneous individual drugs which includemethadone, meprobamate, serotonin, meperidine, amitriptyline,nortriptyline, lidocaine, procaineamide, acetylprocaineamide,propranolol, griseofulvin, valproic acid, butyrophenones,antihistamines, anticholinergic drugs, such as atropine, theirmetabolites and derivatives.

Metabolites related to diseased states include spermine, galactose,phenylpyruvic acid, and porphyrin Type 1.

The next group of drugs is aminoglycosides, such as gentamicin,kanamicin, tobramycin, and amikacin.

Among pesticides of interest are polyhalogenated biphenyls, phosphateesters, thiophosphates, carbamates, polyhalogenated sulfenamides, theirmetabolites and derivatives.

For receptor analytes, the molecular weights will generally range from10,000 to 2×10⁸, more usually from 10,000 to 10⁶. For immunoglobulinsIgA, IgG, IgE and IgM, the molecular weights will generally vary fromabout 160,000 to about 10⁶. Enzymes will normally range from about10,000 to 1,000,000 in molecular weight. Natural receptors vary widely,generally being at least about 25,000 molecular weight and may be 10⁶ orhigher molecular weight, including such materials as avidin, DNA, RNA,thyroxine binding globulin, thyroxine binding prealbumin, transcortin,etc.

Member of a specific binding pair ("sbp member")--one of two differentmolecules having an area on the surface or in a cavity whichspecifically binds to and is thereby defined as complementary with aparticular spatial and polar organization of the other molecule andwould be incapable of specifically binding to the other molecule if itlacked such a spatial and polar organization. The members of thespecific binding pair are referred to as ligand and receptor(antiligand). These will usually be members of an immunological pairsuch as antigen-antibody, although other specific binding pairs such asbiotin-avidin hormones-hormone receptors, nucleic acid duplexes,IgG-protein A, DNA--DNA, DNA--RNA, and the like are not immunologicalpairs but are included in the definition. An analyte can be, and usuallyis, an sbp member.

Ligand--any organic compound for which a receptor naturally exists orcan be prepared.

Receptor ("antiligand")--any compound or composition capable ofrecognizing a particular spatial and polar organization of a molecule,e.g., epitopic or determinant site. Illustrative receptors includenaturally occurring receptors, e.g., thyroxine binding globulin,antibodies, enzymes, Fab fragments, lectins, nucleic acids, protein A,complement component Clq, and the like.

Labeled sbp member--a label, generally capable of electrochemicaldetection or absorption or emission of electromagnetic radiation, acatalyst, frequently an enzyme, bound to an sbp member, The labeled sbpmember is generally a member of the signal producing system.

Antibody--an immunoglobin, or derivative or fragment thereof, having anarea on the surface or in a cavity which specifically binds to and isthereby defined as complementary with a particular spatial and polarorganization of another molecule. The antibody can be monoclonal orpolyclonal and can be prepared by techniques that are well known in theart such as, for example, immunization of a host and collection of seraor hybrid cell line technology.

Antibody for the analyte--an antibody specific for an analyte.

Analog analyte--a modified analyte or analyte analog or surrogate whichcan compete with the analogous analyte in binding to an sbp member,usually a receptor or antibody, the modification providing means to jointhe analyte analog to a label to provide a labeled sbp member. Theanalyte analog will usually differ from the analyte by more thanreplacement of a hydrogen with a bond which links the analyte analog toa hub or label, but need not. The term analyte surrogate refers to acompound having the capability of binding the antibody for the analyte.Thus, the analyte surrogate may bind to the antibody for the analyte ina manner similar to the analyte. On the other hand, the surrogate couldbe, for example, an antibody directed against the idiotype of anantibody to the analyte.

Bibulous member--a porous material having pores of at least 0.1μ,preferably at least 1.0μ, which is susceptible to flow through ortraversal by an aqueous medium in response to capillary force. Suchmaterials are generally hydrophilic or are capable of being renderedhydrophilic and preferably are cellulosic materials and materialsderived from cellulose, such as fiber containing papers, e.g., filterpaper, chromatographic paper, etc., but may include inorganic powderssuch as silica, magnesium sulfate, and alumina; and other naturalpolymeric materials, and synthetic or modified naturally occurringpolymers, such as nitrocellulose, cellulose acetate, poly (vinylchloride), polyacrylamide, cross linked dextran, agarose, polyacrylate,etc.; either used by themselves or in conjunction with other materials;ceramic materials; and the like. The bibulous member can be attached toa support. On the other hand, the bibulous member may provide its ownsupport. The bibulous member may be polyfunctional or be capable ofbeing polyfunctionalized to permit covalent bonding of reagents such asreceptors, antibodies, chelating agents, oxidants, reductants, and thelike, as well as to permit bonding of other compounds which form a partof the signal producing system.

Binding of receptors ligands, and detection agents to the bibulousmember may be accomplished by well-known techniques, commonly availablein the literature. See, for example, "Immobilized Enzymes," IchiroChibata, Halsted Press, New York (1978) and Cuatrecasas, J. Biol. Chem.,245:3059 (1970).

The piece of bibulous material comprising the bibulous member can be asingle structure such as a sheet cut into strips or it can be severalstrips or particulate material bound to a support or solid surface suchas found, for example, in thin-layer chromatography and may have anabsorbent pad either as an integral part or in liquid contact. Thebibulous member can be comprised of several segments in liquid receivingrelationship and preferably bound to a support. The bibulous member canalso be a sheet having lanes thereon or capable of spotting to inducelane formulation, wherein a separate assay can be conducted in eachlane.

The bibulous member can have a rectangular, circular, oval, triagonal orother shape provided that there is at least one direction of flowthrough or traversal of an aqueous medium by capillary migration. Otherdirections of traversal may occur such as in an oval or circular piececontacted in the center with the test solution. However, the mainconsideration is that there be at least one direction of flow. In thefollowing discussion strips of bibulous material will be described byway of illustration and not limitation.

The support for the bibulous member, where a support is desired ornecessary, will normally be water insoluble, non-porous, and rigid andusually will be of the same length and width as the bibulous member butmay be larger or smaller. A wide variety of organic and inorganicmaterials, both natural and synthetic, and combinations thereof, may beemployed provided only that the support does not interfere with thecapillary action of the bibulous member, or non-specifically bind assaycomponents, or interfere with the signal producing system. Illustrativepolymers include polyethylene, polypropylene, poly(4-methylbutene),polystyrene, polymethacrylate, poly(ethylene terephthalate), nylon,poly(vinyl butyrate), glass, ceramics, metals, and the like.

Absorbent member--any porous, hydrophilic bibulous material such asporous polymers, e.g. Porex®, etc., paper, sponge, felt, and the likecapable of absorbing liquid utilized in the method of the presentinvention. The absorbent member is preferably a non-swellable porouspolymer. The absorbent member provides a plurality of functions. Thefirst function is to serve as a receptacle or storage area for the fluidimbibed by the first bibulous member. A second function, which can beserved but need not, is to control the rate at which the fluid traversesthe first bibulous member. Where the absorbent member has a smalldimension or is of a different material from the first bibulous member,it can act to control the rate at which fluid passes through the firstbibulous member. By having irregular dimensions, it can further act toprovide for different rates depending upon whether the sample or reagentsolutions are being imbibed.

A further function for the absorbent member can be to measure the amountof liquid which is imbibed. Preferably, the absorbent member will takeup a defined volume of liquid. By providing for graduations atsequential positions extending away from the first bibulous members andalong the absorbent member, one can determine when the fluid front is ata certain position and removed the device from the medium.Alternatively, one can provide for dyes which will become colored upondissolution in or contact with the fluid front to provide a clear signalthat the device should be removed. For example, pH indicators could beused. Any technique which provides a clear indication of the presence ofthe fluid front can be employed.

In a strip format, the absorbent member will generally have a length ofat least about 1.5, usually 2 cm and not more than about 30 cm, usuallynot more than about 20 cm. The width may vary from about 0.1 mm to about3 cm. These dimensions are primarily for control of the rate and amountof solution imbibed and for convenience in handling and providing forease of observation of the solvent front. The thickness will generallybe about 0.1 mm to 5 mm, usually from about 0.5 to 3 mm. In a padformat, the absorbent member will generally have a thickness of 0.5 to10 mm and an area of 25 to 150 mm².

The absorbent member may be partially or substantially completelyenclosed in a protective casing, conveniently a clear or partially, orin some situations completely, opaque enclosure. The absorbent membershould not directly contact the sample solution. It will usually benecessary that at least a minimum and normally a specific volume ofsolution containing an analyte pass through the first bibulous member.This can be best achieved by avoiding substantial contact of theabsorbent member with the assay sample solution.

Depending upon the particular protocols involved and the construction ofthe device, the enclosure may be removable or irremovable, may encaseonly the absorbent member or may encase additionally the first andsecond bibulous members, may provide for one or more windows and willnormally be of a sturdy inert impermeable material which will providemechanical protection for the bibulous material and will not interferewith the performance of the assay. Normally an air opening will beprovided to prevent the entrapment of air within the enclosure.

Label--A label may be any molecule bound to an sbp member that isrequired to produce a signal. In the subject invention, the label may beinert and serve solely as a binding site for a member of the signalproducing means or it may spontaneously produce a detectable signal ormay produce a detectable signal in conjunction with a signal producingmeans. The label may be isotopic or nonisotopic, preferably nonisotopic.However, an isotopic label can be preferred for achieving highsensitivity when using radio-autographic detections with photographicfilm.

Signal producing means--means capable of interacting with the label toproduce a detectible signal. Such means include, for example,electromagnetic radiation, heat, chemical reagents, and the like. Wherechemical reagents are employed, some of the chemical reagents can beincluded as part of a developer solution. The chemical reagents caninclude substrates, coenzymes, enhancers, second enzymes, activators,cofactors, inhibitors, scavengers, metal ions, specific bindingsubstances required for binding of signal generating substances, and thelike. Some of the chemical reagents such as coenzymes, substances thatreact with enzymic products, other enzymes and catalysts, and the likecan be bound to the strip.

Signal producing system--The signal producing system may have one ormore components. The signal producing system includes all of thereagents required to produce a measurable signal which may include alabel and signal producing means capable of interacting with the labelto produce a signal.

The signal producing system provides a signal detectable by externalmeans, normally by measurement of electromagnetic radiation, desirablyby visual examination. For the most part, the signal producing systemincludes a chromophoric substrate and enzyme, where chromophoricsubstrates are enzymatically converted to dyes which absorb light in theultraviolet or visible region, phosphors or fluorescers, or chromophoricindicators such as chelating agents, redox indicators, pH indicators, orthe like.

The signal-producing system can include at least one catalyst as alabel, usually an enzyme, and at least one substrate and may include twoor more catalysts and a plurality of substrates, and may include acombination of enzymes, where the substrate of one enzyme is the productof the other enzyme. The operation of the signal producing system is toproduce a product which provides a detectable signal in relation to thepresence or amount of analyte in a sample.

Two catalysts may be employed, either a combination of an enzyme and anon-enzyme catalyst or two enzymes, where the two catalysts are relatedin that the product of one is the substrate of the other. In thissystem, there need be only one substrate which can undergo successivechanges catalyzed by the catalysts, which results in the compoundinvolved with production of a detectable signal. For the most part,however, there will normally be a substrate for the first enzyme in theseries and a second compound, which serves as a precursor to thecompound involved in the production of the signal, normally providingthe compound which produces the signal. Thus, the product of the firstenzyme may react with the precursor to the compound that produces asignal to provide the compounds that generates the signal.

Where two enzymes are employed, the involved reactions will be, for themost part, hydrolysis or redox reactions. In the case of hydrolysis, aderivatized dye precursor that has a hydrolytically labile bond, thehydrolytic enzyme and an enzyme that catalyzes the conversion of thereleased dye precursors to a dye conversion product is illustrative ofthis type of system. In redox reactions, a first enzyme can produce anessential oxidizing substrate required for the second enzyme, where thesecond enzyme catalyzes the reaction between the oxidizing substrate anda dye precursor.

Where two enzymes are used, the first enzymatic reaction may involvehydrolytic cleavage or a redox reaction of the substrate to provide aproduct which is the substrate of another enzyme. The first situationmay be illustrated by glucose-6-phosphate being catalytically hydrolyzedby alkaline phosphatase to glucose, where glucose is a substrate forglucose oxidase. The second situation may be illustrated by glucosebeing oxidized by glucose oxidase to provide hydrogen peroxide whichwould enzymatically react with a leuco dye to produce a signalgenerator.

Coupled catalysts can also involve an enzyme with a non-enzymaticcatalyst. The enzyme can produce a reactant which undergoes a reactioncatalyzed by the non-enzymatic catalyst or the non-enzymatic catalystmay produce a substrate (includes coenzymes) for the enzyme. A widevariety of non-enzymatic catalysts which may be employed are found inU.S. Pat. No. 4,160,645, issued Jul. 10, 1979, the appropriate portionsof which are incorporated herein by reference.

Various combinations of enzymes may be employed to provide a signalgenerating compound. Particularly, combinations of hydrolases may beemployed to produce an insoluble signal generator, Alternatively,combinations of hydrolases and oxidoreductases can provide the signalgenerating compound. Also, combinations of oxidoreductases may be usedto produce an insoluble signal generating compound.

For combinations of enzymes one enzyme can be non-diffusively bound tothe bibulous material, either the first or second bibulous members,while the other enzyme can be a label conjugated to the analyte.Additionally, one or more other members of the signal producing systemcan be bound to the bibulous material depending on the particular signalproducing system chosen or the particular protocol followed.

In some assays in order to have a detectable signal, it is necessary toprovide means for amplifying the signal member produced by the presenceof a label at a zone on a bibulous situs. Therefore, it will usually bepreferable for the label to be a catalyst or luminescent compound orradioisotope, most preferably a catalyst. Preferably, catalysts areenzymes and coenzymes which can produce a multiplicity of signalgenerating molecules from a single label.

An enzyme or coenzyme is employed which provides the desiredamplification by producing a product, which absorbs light, e.g., a dye,or emits light upon irradiation, e.g., a fluorescer. Alternatively, thecatalytic reaction can lead to direct light emission, e.g.,chemiluminescence. A large number of enzymes and coenzymes for providingsuch products are indicated in U.S. Pat. No. 4,275,149 bridging columns19 to 23, and U.S. Pat. No. 4,318,980, columns 10 to 14, whichdisclosures are incorporated herein by reference.

A number of enzyme combinations are set forth in U.S. Pat. No.4,275,149, bridging columns 23 to 28, which combinations can find use inthe subject invention. This disclosure is incorporated herein byreference.

Of particular interest are enzymes which involve the production ofhydrogen peroxide and the use of the hydrogen peroxide to oxidize a dyeprecursor to a dye. Particular combinations include saccharide oxidases,e.g., glucose and galactose oxidase, or heterocyclic oxidases, such asuricase and xanthine oxidase, coupled with an enzyme which employs thehydrogen peroxide to oxidize a dye precursor, that is, a peroxidase suchas horseradish peroxidase, lactoperoxidase, or microperoxidase.Additional enzyme combinations may be found in the subject matterincorporated by reference. When a single enzyme is used as a label,other enzymes may find use such as hydrolases, transferases, andoxidoreductases, preferably, hydrolases such as alkaline phosphatase andβ-galactosidase. Alternatively luciferases may be used such as fireflyluciferase and bacterial luciferase.

Illustrative coenzymes which find use include NAD[H]; NADP[H], pyridoxalphosphate; FAD[H]; FMN[H], etc., usually coenzymes involving cyclingreactions, see particularly U.S. Pat. No. 4,318,980.

The product of the enzyme reaction will usually be a dye or fluorescer.A large number of illustrative fluorescers are indicated in U.S. Pat.No. 4,275,149, columns 30 and 31, which disclosure is incorporatedherein by reference.

Liquid receiving relationship--Two bibulous members are in liquidreceiving relationship when liquid can pass from one bibulous member tothe other. This relationship can be either direct or indirect. A directliquid receiving relationship can be realized by having two members incontact with one another so that liquid from one member will pass bycapillary action to the other member and can be drawn from one member toanother. An indirect liquid receiving relationship can be realized byhaving the two members in close proximity such that liquid can pass fromone to the other. The two members can be separated by porous spacersthat are inert and substantially non-resistant to fluid flow. On theother hand, or in conjunction with porous spacers, fluid flow-resistantmembers can be employed to control the rate of fluid flow between twomembers. Spatial orientation of the two members may be utilized toachieve a liquid receiving relationship.

Immunochromatograph--The immunochromatograph has a sbp member, eitherligand or receptor, bound in a region to a bibulous support which allowsfor the movement of a liquid by capillarity across the region withtransport of the analyte and, as appropriate, any members of the signalproducing system. The sbp member is non-diffusively bound to thesupport, either covalently or non-covalently. The area to which the sbpmember is uniformly bound is referred to as the "immunosorbing zone." Inaddition, one or more members of the signal producing system can benon-diffusively bound to the bibulous support, either covalently ornon-covalently. Furthermore, a reactant for a label such as an enzymesubstrate can be bound to the support.

Component--a molecule of interest, the amount thereof is to bedetermined in accordance with one aspect of the present invention. Thecomponent can be an inorganic compound such as an inorganic ion, eithercation or anion, for example, metal ions including alkali metal ionssuch as lithium, potassium, sodium, etc., alkaline earth metal ions suchas magnesium, calcium and so forth, copper, cobalt, manganese, aluminum,iron (ferrous or ferric), chromium, nickel, zinc, cadmium and so forth,and non-metal ions such as halides (chloride, bromide, fluoride, iodide,etc.), oxides, sulfides, sulfates, phosphites, phosphates, selenides,oxidized halogens such as iodates, periodates, chlorates, chlorites, andso forth. In general, any inorganic molecule for which there exists aninterreactive reagent and which is spectroscopically detectible,preferably by reaction with a compound that can react with the inorganicmolecule to provide a spectroscopic change that can be quantitated inaccordance with the present invention.

The component may also be an organic molecule for which there exists aninterreactive reagent such as a binding partner, for example, an sbpmember, and which is spectroscopically detectible, preferably byreaction with a compound that can react with the organic compound toprovide a spectroscopic change. The component can itself be measured inthe assay or be a compound utilized in an assay for an analyte. Typicalorganic molecule components include enzyme substrates, such as glucose,ethanol, cholesterol, arabinitol, urea, lactates, amino acids,triglycerides, etc.; reducing agents such as ascorbate, uric acid,mercaptans, hydroquinones, etc.; oxidants such as quinones anddisulfides; ligand-label conjugates; receptor-label conjugates; and soforth. It is an important feature of one aspect of the present inventionthat an analyte as well as a component and a reagent interreactive withthe component can all be determined quantitatively.

Reagent interreactive with the component--a substance that is capable ofinterreacting stoichiometrically with a component either chemically orphysically and is bound or is capable of becoming bound to a firstbibulous member zone. Chemical interreaction means generally that thereagent is capable of transforming the component into another substanceby breaking or forming chemical bonds and is thereby itself converted toa new compound. Physical interreaction means generally that the reagentinterreacts stoichiometrically with the component other than chemically.For example, the reagent can be a ligand or receptor that bindsspecifically with the component by formation of hydrogen bonds,electrostatic interactions, hydrophobic interactions and so forth. Thereagent can also be an inorganic or organic molecule. Exemplary ofinorganic molecules are metallic and non-metallic ions, oxidants andreductants, transition metal halides, organomercury halides, etc.Exemplary of organic molecules are chelating agents, sbp members,diazonium salts, quinones, iodoso compounds, disulfides, iodoacetamides,hydroquinones, hydroxyanilines, benzidines, hydrazides, paraquat, etc.

The interreaction can involve oxidation or reduction such as, forexample, the oxidation or reduction of inorganic molecules, directcovalent bond formation, or coordinate metal-ligand bond formation,including chelation, electrophilic or nucleophilic substitution,cycloaddition, etc.

Of different composition than the first zone--the composition of thesecond bibulous member or the second zone should be different from thatof the first bibulous member or the first zone. The following is adescription, by way of illustration and not limitation, of situations inwhich different composition can be realized. Other situations will besuggested to those skilled in the art. One such example occurs where thesecond bibulous member does not have bound to it any substanceinterreactive with a compound or an analyte. Another example occurswhere the second bibulous member contains a substance interreactive witha component or a product produced upon reaction of a component with thereagent, which substance is different from the reagent on the firstbibulous member. Another example is a situation where the reagent of thefirst bibulous member is also found on the second member but theconcentrations of each on the members is different.

Ancillary Materials--Various ancillary materials will frequently beemployed in the assay in accordance with the present invention. Forexample, buffers will normally be present in the assay medium, as wellas stabilizers. Frequently, in addition to these additives, additionalproteins may be included, such as albumins, or surfactants,particularly, non-ionic surfactants, binding enhancers, e.g.polyalkylene glycols, or the like.

As mentioned above, one method in accordance with the present inventionfor conducting an assay provides in combination a first bibulous memberzone ("first zone") and a liquid medium containing a component. Thefirst zone has non-diffusively bound thereto a reagent interreactivewith said component. Conditions are chosen wherein the liquid mediumtraverses all of the first zone and at least a portion of the componentcontained therein reacts with all of the reagent and therefore alsotraverses all of the first zone and is transported by capillarymigration of the liquid medium into a second bibulous member zone("second zone") of different composition than the first zone bycapillary migration. The distance the component migrates into the secondzone or the difference in the distances the liquid medium and thecomponent migrate into the second zone is determined. The distance orthe difference is related to the amount of the component in the liquidmedium or the amount of the reagent. When it is desired to determine theamount of one of these substances the amount of the other substanceswill be predetermined and the distance of migration will be related tothe distances obtained with known calibrators. The distances ofmigration can be detected by contacting the second zone with anappropriate detection agent, by the presence of an appropriate detectionagent on, or bound to, the second zone that will produce a detectiblesignal on the second zone in relation to the presence of the component,or by direct detection of the component on the second zone.

The assay may be configured to measure the component concentration orthe reagent concentration.

As described above, prior to the combination of the liquid medium andthe first zone the reagent can be caused to non-diffusively bind to thefirst zone by contacting the first zone with a solution containing thereagent. The difference or the distance as defined above can then berelated to the amount of the reagent in the solution. Furthermore, theamount of the reagent can then be related to the amount of an analyte ina solution of the reagent.

A novel feature of this aspect of the invention lies in the manner inwhich breakthrough volume as mentioned earlier is measured. The liquidwhich has passed through the first zone enters a second zone in whichthe volume taken up is related to the distance of liquid migration alongthe zone. After a suitable volume of liquid has entered the second zone,the border demarking the region containing the component and the regionlacking a significant concentration of the component are located.Measuring the distance along the zone which liquid has migrated and thedistance along the zone which the component has migrated as demarked bythe border allows determination of the breakthrough volume. If thelength of the second zone is the same for all assays, and the volumepassed through the first zone just fills the second zone, thenmeasurement of the migration distance, that is, the distance to theborder, is sufficient for a determination of the breakthrough volume.

The distance of migration can be determined by direct detection of thecomponent on the second zone or by contacting the second zone with anappropriate detection agent when said detection agent is not alreadypresent, which detection agent will produce a detectible signal on thesecond zone in relation to the presence of the component. The detectionagent can be a substance that chemically interacts with the componentusually to produce a visual signal such as color. The detection agentcan be a member of a signal producing system. In general, detectionagents will be reactive with an assay component or the product ofreaction of an assay component and a reagent, and on reaction willproduce a spectroscopically detectible change. Typical detection agentsare chelators such as phenanthrolines, caroboxypyridones, phenols,β-diketones, etc.; oxidants such as quinones, ceric ion, manganesedioxide, periodates, chromates, disulfides, tetrazolium salts, etc.;reductants such as leuco dyes, mercaptans, thiosulfates, hydroquinones,benzidines, etc.; catalysts such as enzymes, co-enzymes, prostheticgroups, Medola blue, etc.; enzyme substrates; and so forth.

The first zone and the second zone can be integral to a single bibulousmember, provided that the two zones must be related such that the liquidmedium traverses the first zone prior to contacting the second zone andthe two zones must have different compositions. On the other hand, thefirst zone can be integral to a first bibulous member and the secondzone can be integral to a second bibulous member. The first zone canthen, for example, be brought into liquid receiving relationship withthe second zone prior to providing the combination of the first zone andthe liquid medium. A convenient device for this aspect of the presentinvention is depicted in FIG. 1.

The component and the reagent can each be a member of the same or adifferent specific binding pair. For example, the component can be anenzyme labeled ligand analog and the reagent can be antibody for theligand. The method can also include determination of the amount of ananalyte which is a ligand.

As mentioned above, the component and the reagent can be chemicallyinterreactive or physically interreactive. The method can be applied tothe determination of both inorganic and organic molecules.

Another approach in this quantitative aspect of the present inventioninvolves contacting a first bibulous member zone ("first zone") with aliquid medium containing a component. The first zone has non-diffusivelybound therein a reagent interreactive with the component. Contactingoccurs under conditions wherein the liquid medium and at least a portionof the component contained therein traverse all of the first zone andmigrate into a second bibulous member zone ("second zone") of differentcomposition than the first zone by capillary migration. Measuring thedistance the component has migrated into said second zone or thedifference in the distances the medium and the component have migratedinto the second zone, allows the determination of the amount of thecomponent in the liquid medium.

Another approach in accordance with this aspect of the inventioninvolves combining a solution of an analyte with a first bibulous memberwherein a reagent capable of reacting with the analyte is or becomesnon-diffusively bound to the bibulous member. Next, a liquid mediumcontaining a component is combined with the first bibulous memberwherein the component is interreactive with the reagent. The firstbibulous member is in liquid receiving relationship with a secondbibulous member. Conditions are chosen wherein the liquid mediumtraverses the first bibulous member and at least a portion of the secondbibulous member by capillary action. Determination of a border on thesecond bibulous member is made. The position of the border is related tothe amount of analyte in the solution or the amount of reagent on thefirst bibulous member or the amount of the component in the liquidmedium. By way of example, the analyte can be a drug, the reagent can bean antibody for the drug, and the component can be an enzyme labeleddrug analog.

In another approach a combination is provided in a first liquid medium.The combination comprises (1) a first bibulous member and (2) ananalyte, wherein a reagent for the analyte is present in the medium oron the first bibulous member. The first bibulous member is in liquidreceiving relationship with an absorbent member. Conditions are chosenwherein the first medium flows through the first bibulous member and atleast a portion of the absorbent member by capillary action and thereagent and usually the analyte become non-diffusively bound to thebibulous member. Thus, the first liquid medium can be contacted with allof the first member at once to achieve flow through or the first liquidmedium can be contacted with only a portion of the first bibulousmember. In the latter case the medium will traverse the remainder of themember by capillary action. The liquid receiving relationship betweenthe bibulous member and the absorbent member is then terminated. Acombination is provided, in a second liquid medium, comprising a portionof the first bibulous member and a component interreactive with thereagent, the first bibulous member being in liquid receivingrelationship with a second bibulous member. Conditions are chosen suchthat the second medium traverses the first bibulous member and at leasta portion of the second bibulous member by capillary action, with theproviso that the first bibulous member is in liquid receivingrelationship with the absorbent member and the second bibulous member atsubstantially different times. Next, a border is determined on thesecond bibulous member. The position of the border is related to theamount of analyte in the first medium, or the amount of reagent, or theamount of the component in the second liquid medium.

In another aspect of the present invention the method comprises causinga reagent such as an sbp member in a first liquid medium or solution tobecome bound to a first bibulous member by contacting the first bibulousmember with the medium. The first bibulous member is in liquid receivingrelationship with an absorbent member. Conditions are chosen such thatthe first medium traverses or flows through the first bibulous memberand at least a portion of the absorbent member by capillary action. Thefunction of the first bibulous member is to bind the reagentquantitatively and tightly. The reagent must be bound in a defined orpredetermined amount depending on the predetermined minimum detectibleamount of analyte of interest. In general, the reagent bound will be nomore than 20 fold less or 20 fold greater than the amount of analyte orcomponent that traverses the first bibulous member. Frequently, all ofthe reagent in the first medium or solution will become bound.

In one approach in the present invention, a combination is provided in afirst liquid, usually aqueous, medium or solution. The combinationincludes (1) a first bibulous member, a portion of which has a firstreagent such as an sbp member non-diffusely bound to it, and (2) asample suspected of containing an analyte. When the analyte is notcapable of binding to the first reagent, a second reagent capable ofbinding to the analyte and to first reagent is present in the medium oron the first bibulous member.

A combination can be formed in one of a number of orders. For example,the first bibulous member can have the first reagent bound thereto. Aportion of the first bibulous member ("contact portion") is contactedwith the first medium or solution containing the analyte. Where thesample is an aqueous medium containing the analyte, the sample canaccount for some or all of the first medium or solution. The primaryconsideration is that a combination be provided, in a first medium, ofthe sample and the first reagent on a portion of a first bibulousmember, for example, a strip, and either flowing through or traversingthe member through capillary action. This movement can be upward,downward, horizontal, or combinations thereof. The movement of the firstmedium along or through the first bibulous member can be driven by theabsorbent member in liquid receiving relationship with the firstbibulous member. Such a driving or wicking type action provides forbetter control of the liquid contacting the chemically active part ofthe first bibulous member.

When the analyte is not capable of binding to the first reagent, asecond reagent capable of binding to the analyte (as distinguished frombeing already bound to the analyte) and the first reagent is present inthe first medium or solution or on the first bibulous member. If thesecond reagent is on the first bibulous member, it is generallydiffusively bound so that contact with the aqueous medium will allow thesecond reagent to diffuse into the first medium. This diffusion can takeplace during the movement along the first bibulous member by the firstmedium.

The second reagent, as mentioned above, has the characteristic ofbinding to the analyte and to the first reagent. Where the analyte andthe first reagent are the same or analogous, the second reagent can be abinding partner, such as an sbp member, for the analyte or the analyteanalog. However, the first sbp member can be other than an analyte or ananalyte analog. The function of the first reagent is to either bind theanalyte or a second reagent in relation to the amount of analyte in thesample. Accordingly, where the first reagent does not bind the analyteor an analyte analog, the first reagent can be chosen to be a bindingpartner to the second reagent. In this circumstance, the second reagentwill have two parts, one part, for example, a second sbp member, capableof binding to the analyte, and one part, for example, a third sbpmember, capable of binding to the first reagent. The part capable ofbinding to the analyte can be complementary to the analyte such as, forexample, an sbp member for the analyte, such as an antibody. The otherpart of the second reagent can be a small organic molecule of amolecular weight less than 1500, and preferably greater than 250. Thefirst reagent in this situation can be an sbp member that iscomplementary to either the part complementary to the analyte or thesmall organic molecule. Thus, the first reagent can be, for example, anantibody for an antibody, or an antibody for the small organic molecule.Exemplary small organic molecules are biotin, fluorescein, LSD,morphine, benzoylecgonine, and the like.

The amount of the second reagent that becomes bound to the bibulousmember is related to the presence in the sample of the correspondinganalyte in an amount at or exceeding a predetermined minimum detectibleamount of that analyte. This minimum detectible amount is generally thatamount above which the analyte is considered to be present. For example,for a drug one may be interested in only whether the drug is present ina certain concentration range. Although the drug might be present belowthat range in the sample or test solution, it would not be consideredpresent because its concentration is not at or above the minimumdetectible amount.

The aqueous medium or solution and the liquid medium may be up to about40 weight percent of other polar solvents, particularly oxygenatedsolvents of from 1 to 6, more usually of from 1 to 4 carbon atoms,including alcohols, ethers and the like. Usually, the cosolvents will bepresent in less than about 20 weight percent. Under some circumstancesdepending on the nature of the sample, some or all of the aqueous mediumcould be provided by the sample itself. Under certain circumstances, anon-aqueous solution or liquid medium can be employed using a polar ornon-polar organic solvent. Such a circumstance can be, for example,where water is not necessary for reaction of the materials in an assayor where water would be detrimental to the activity of such materials.

When the aqueous medium is used, the pH for the medium will usually bein the range of 4-11, more usually 5-10, and preferably in the range ofabout 6-9. The pH is chosen that will facilitate the interractioninvolved in the assay such as, for example, to maintain a significantlevel of binding affinity of the binding members and optimal generationof signal by the signal producing system. Various buffers may be used toachieve the desired pH and maintain the pH during the assay.Illustrative buffers include borate, phosphate, carbonate, tris,barbital and the like. The particular buffer employed is not critical,but in individual assays, one buffer may be preferred over another.

It may be desirable in some instances to include from about 0.05 to 0.5weight percent of a non-ionic detergent with the sample. Variouspolyoxyalkylene compounds may be employed of from about 200 to 20,000daltons.

Moderate, and desirably substantially constant, temperatures arenormally employed for carrying out the assay. The temperatures for theassay and production of a detectable signal will generally be in therange of about 4°-50° C., more usually in the range of about 10°-40° C.,and frequently will be ambient temperatures, that is, about 15°-25° C.

The concentration in the first aqueous medium or solution of thecomponent or the analyte that may be assayed will generally vary fromabout 10⁻⁴ to about 10⁻¹⁵ M, more usually from about 10⁻⁶ to 10⁻¹⁴ M.Considerations, such as the concentration of the component or analyte ofinterest and the protocol will normally determine the concentration ofthe other reagents.

While the concentrations of many of the various reagents in the sampleand reagent solutions will generally be determined by the concentrationrange of interest of the component and/or the analyte, the finalconcentration of at least some of the reagents will normally bedetermined empirically to optimize the sensitivity of the assay over therange of interest. With certain protocols, individual reagents may beused in substantial excess without detrimentally affecting thesensitivity of the assay.

When a strip is employed as the first bibulous member, the size of thestrip is dependent on several considerations. The primary considerationis to capture a sufficient amount of analyte or reagent or component sothat a sufficient signal will subsequently be obtained to achieve asensitive and accurate assay. In that respect, the qualitative aspect ofthe present invention provides for increased sensitivity because analyteor reagent can be concentrated on a first bibulous member to fill allavailable binding sites by flowing a medium through such member into anabsorbent member prior to bringing the first member into liquidreserving relationship with the second bibulous member. In general, forupward flow strips the fluid retention volume will be usually greaterthan 1 μL, preferably at least 5-200 μL. For downward flow stripsretention volumes as low as 1-20 μL can be used but volumes of 5-200 μLare preferable.

Thickness of the strips is not critical and will normally be 0.05-2 mm,usually 0.1-1 mm, preferably 0.2-0.7 mm. Generally the minimum thicknessis dictated by the strength of the material and the need to produce areadily detectible signal whereas the maximum width will be dictated byconvenience of handling and cost of the reagents.

To permit conservation of reagents and provide for samples of limitedsize, the width of the strip will generally be relatively narrow,usually less than 20 mm, preferably less than 10 mm. Generally, thewidth of the strip will not be less than about 1.0 mm and will usuallyrange from about 2 mm to 12 mm, preferably from about 4 mm to 8 mm.

The length of the strip will depend on the concentration of the analyteor the component or the reagent and practical considerations such asease of handling and the number of situses on the strip and will beabout 0.5 cm to 40 cm, usually about 1 cm to 25 cm, preferably about 4to 20 cm but may be of any practical length. The structure of the stripcan be varied widely and includes fine, medium fine, medium, mediumcoarse and coarse. In general, smaller pore size and finer material willprovide slow capillary flow and efficient capture of the desired reagenton the strip. Courser more porous materials provide faster flow, but theefficiency of capture is reduced. Selection of the porosity of thematerial depends on the rate of binding of the components for a givenassay. Preferably, the pore size should not be so small as to restrictflow and not be so large as to occlude serum, if serum is the sample ofinterest. Such a situation allows for one of the benefits of the presentinvention, namely, avoiding the necessity of a washing step.

The cross-sectional dimensions of the strip have been described in thepreceding discussion in terms of a rectangle for the purpose ofillustration and not limitation. As mentioned above, other shapes suchas circular, triagonal, oval, etc, fall equally within the scope of thisinvention. The dimensions thereof can be determined by those skilled inthe art with reference to the disclosure herein.

Other reagents, which are members of the detection system such as, e.g.,the signal producing system, can vary widely in concentration dependingupon the particular protocol and their role in signal production.Usually, the amount of a reagent, e.g., first sbp member, on the firstbibulous member will be homogeneously or uniformly bound on at least aportion thereof between the contact portion and the absorbent member andis determined with reference to the predetermined minimum detectibleamount of component or analyte. Usually, this amount will not exceed 10⁴times the maximum amount of component or analyte to be assayed and willnot be less than about equal the minimum amount of the reagent such asthe first reagent used in the assay. The minimum concentration of thereagent is determined with reference to the minimum detectible amount ofthe component. This amount will depend on the sensitivity of detectionand may vary from 10⁻¹⁹ mole to 10⁻⁶ mole, usually 10⁻¹⁸ to 10⁻¹⁰ mole.Where the first reagent is employed to capture a second reagent, theamount of first reagent on the first bibulous member can be chosen suchthat only a predetermined amount of second reagent will become bound.

In carrying out an assay for an analyte, the protocol will usuallyinvolve forming a combination in an aqueous medium of the samplesuspected of containing the analyte and the first reagent. The samplemay be derived from a wide variety of sources, such as physiologicfluids, illustrated by saliva, blood, serum, plasma, urine, ocular lensfluid, spinal fluid, etc., food products such as milk and wine, chemicalprocessing streams, food waste water, etc.

The contact portion of, or the entire, first bibulous member iscontacted with the first medium, usually by immersion such as by dippingof the contact portion into the medium. However, contact of the firstbibulous member with the first liquid medium or solution can be carriedout by other techniques such as by spotting the first medium on thefirst bibulous member. This technique has particular application to afirst bibulous member that is strip-like, circular, oval, sheet-like,etc. Wetting of the first bibulous member by capillary action usually isallowed to continue until a defined volume of medium has traversed thebibulous member. Commonly, at least 20 μL, usually at least 50 μL,frequently at least 100 μL, of the first medium will traverse thebibulous member. The upward limit of first medium traversing thebibulous member is generally determined by practical considerations suchas the size of the device, and the like.

As a practical matter, relatively short times are desired for the firstmedium to traverse the first bibulous member. Usually, the traverse ofthe first medium over the first bibulous member will take at least 30sec and not more than 1 hour, more usually from about 1 min to 30minutes, but periods as long as 24 hours can be used.

After the first medium has traversed the first bibulous member and atleast a portion of the absorbent member, or after contact of the entirefirst bibulous member with the first medium, the first bibulous memberis brought into liquid receiving relationship with a second bibulousmember. In a preferred embodiment the first bibulous member is in liquidreceiving relationship with the absorbent member and a chromatographicmember that will allow liquid flow by capillary action, such as theimmunochromatographic member, at substantially different times. Morepreferably, the liquid receiving relationship between the first bibulousmember and the absorbent member is terminated prior to instituting theliquid receiving relationship between the second bibulous member and thesecond medium. The relationship between the first bibulous member andthe absorbent member on the one hand and the second bibulous member onthe other hand may be instituted or terminated by physical or mechanicalmeans or a combination thereof.

A portion of the second bibulous member is contacted with a secondliquid, usually aqueous, medium. The second medium traverses the firstbibulous member and at least a portion of the second bibulous member bycapillary action. A component in the second medium becomesnon-diffusively bound to the second bibulous member in relation to thepresence of analyte in the first medium. The second bibulous member mayhave bound to it a detection agent.

The methods of the present invention can provide either a qualitativeresult or a quantitative determination of an analyte, a reagent, or acomponent. For a qualitative result in the present invention, a liquidreceiving relationship between the first bibulous member and the secondbibulous member is established only after the component or analyte hasbeen concentrated on the first bibulous member. For a quantitativedetermination, the second bibulous member can be a chromatographicmember such as an immunochromatographic member. In one embodiment wherea reagent is employed, the component can be, for example, a conjugate ofan enzyme and an analyte analog. Depending on the presence or amount ofanalyte in the sample, the component will become bound to reagentnon-diffusively bound to the first bibulous member. If analyte ispresent in the sample above a minimum detectible amount, the amount ofreagent is chosen such that a proportional amount of component will notbind to the first bibulous member but rather traverse to the secondbibulous member. In some instances the second bibulous member maycontain a binding partner for the component. In the instance where thecomponent is an enzyme labeled analyte analog, this binding partner maybind to the enzyme portion of the conjugate or to the analyte analogportion of the conjugate. Accordingly, this binding partner can be anantibody for the enzyme or an antibody for the analyte analog. Thecomponent can include a label, or be capable of binding to a label. Thebinding partner on the second bibulous member can be a receptor for anantibody, for example, protein A in the circumstance where the firstreagent is an antigen specific for immunoglobulin analyte.

Examples of immunochromatographic methods are described in U.S. Pat.Nos. 4,168,146 and 4,435,504, the disclosures of which are incorporatedherein by reference in their entirety. As applied to the presentinvention, the component in the second aqueous medium will traverse theimmunosorbing zone of the immunochromatographic member, being carriedalong with the liquid from the first bibulous member. The componentbecomes bound to the second bibulous member through, for example, theintermediacy of sbp member complex formation. For a qualitative resultthe immunosorbing zone can be a narrow band or small situs, which willprovide an indication of the presence or absence of the analyte in thesample. The portion of the second bibulous member traversed by thesecond medium depends on the particular protocol involved but willpreferably be the entire bibulous member. However, as a general rule,for both qualitative and quantitative determinations, the medium willtraverse the portion of the second bibulous member at least through theimmunosorbing zone. The dimensions of the second bibulous member can bedetermined according to the same considerations as those described abovefor the first bibulous member. The relationship of the size of theimmunosorbing zone to the remainder of the second bibulous member issuch that a sufficient amount of component can be passed through theimmunosorbing zone in order to realize an accurate assay.

In a qualitative assay, following the traversal of the first and secondbibulous members by the second medium, the presence of the component onor bound to the second bibulous member is determined. The presence ofthe component on the second bibulous member is related to the amount ofanalyte in the sample. Where the component contains a label, the secondbibulous member can be examined for the presence of a detectible signal.If the signal is the result of a radioactive label, or the like, thesignal can be detected directly at the second bibulous member. Wherechemical agents form part of the signal producing means that includesthe label, the contact portion of the first bibulous member can bedipped into, or otherwise contacted with, a developer solutioncontaining remaining members of the signal producing system. Where onlya part of the second bibulous member is contacted, this developersolution is allowed to flow along the first bibulous member through thesecond bibulous member. Depending on the protocol, a washing step may ormay not be utilized. Alternatively, the second bibulous member can becontacted directly with the developer solution, such as by totaldipping, spraying, and the like.

Depending on the protocol, it may be desirable to first contact thesecond bibulous member with a developer solution and then subsequentlycontact the second bibulous member with any remaining members of thesignal producing system not present in the developer solution or thefirst and second mediums or present on the first or second bibulousmembers.

Generally, a sufficient time is allowed to elapse prior to measuring thesignal to produce an amount of the signal producing compound required todefine the region of the second bibulous member in which the componentis found or bound. Once the detectible signal has been produced, thepresence or absence of the analyte or the amount of analyte in thesample and/or the amount of reagent or component is known. Forquantitative determinations, the signal producing system provides themanner by which the area in the second bibulous member to which theanalyte or component is found or bound may be distinguished from thearea in which it is absent. In this way, the distance from apredetermined point on the chromatograph is a measure of the amount ofanalyte in the sample and/or the component or reagent. In oneembodiment, the region of the second bibulous member traversed by thecomponent is observable due to the production of a detectible signalsubstantially uniformly throughout the region in which the component ispresent.

In one particular embodiment of the present invention, an aqueous mediumis prepared containing a sample suspected of containing an analyte and afirst reagent, which is a conjugate of an antibody for the analyte and asmall organic molecule. The aqueous medium is contacted with a contactportion of a bibulous strip having non-diffusively bound thereto eitheran antibody for the small organic molecule or an antibody thatrecognizes the antibody for the analyte. An end portion of a bibulousstrip is in contact with an absorbent strip. The medium traverses thebibulous strip and part of the absorbent strip. The antibody on thebibulous strip captures the first reagent. If analyte is present in theaqueous medium, the analyte is captured by the antibody of the firstreagent.

The liquid receiving relationship between the bibulous strip and theabsorbent member is terminated and the bibulous strip is brought intoliquid receiving relationship with a second bibulous strip. A secondliquid medium containing a component which is an enzyme labeled analyteanalog is brought into contact with the contact portion of the firstbibulous strip. The second bibulous strip contains antibody that bindsto either the analyte analog or enzyme portion of the second reagent orto the conjugate itself. The antibody on the second bibulous material isnon-diffusively and uniformly bound to a substantial portion of thesecond bibulous strip to form an immunochromatograph. The second aqueousmedium traverses the first bibulous strip and the second bibulous strip.

If analyte is present in the sample, the analyte occupies the bindingsites on the antibody for the analyte that is bound to the firstbibulous strip. Consequently, the enzyme labeled analyte analog does notbind to the occupied binding sites on the first bibulous member and thusmigrates to the second bibulous strip where it is captured by theantibody on the second bibulous strip. The amounts of the first reagentand component employed, therefore, must be predetermined amounts basedon the suspected concentration range of interest of the analyte. In aqualitative assay, if no analyte or an amount of analyte under apredetermined concentration of interest is in the sample, then thecomponent all becomes bound to the first bibulous member so that nocomponent will migrate to the second bibulous strip. However, if analyteis present in the sample, the component migrates to the second bibulousmember in proportion to the amount of analyte in the sample. In aquantitative assay, provided amounts are selected such that at leastsome component will migrate to the second bibulous strip even when noanalyte is present in the sample, the component will migrate a distancealong the second bibulous member in proportion to the amount of analytein the sample.

After the second liquid medium has traversed the first bibulous stripand the second bibulous strip, contact between the first bibulous memberand the second aqueous medium is terminated. The second bibulous stripis then contacted with a developer solution in order to determine thelocation of enzyme conjugate bound to the second bibulous strip. To thisend the first and second bibulous strips can be immersed in a developersolution or the second bibulous strip can be removed from liquidreceiving relationship with the first bibulous strip and contacted withthe developer solution, which contains the remaining members of thesignal producing system. In such a way, the distance of migration of thecomponent on the second bibulous strip is determined and is correlatedwith the amount of analyte in the sample for a quantitative result. Thiscorrelation may be the result of carrying out the above method on knownsamples to determine migration distances and comparing the migrationdistance in the unknown sample with known values in order to determinethe amount of analyte present in the sample.

The developer solution can contain the remaining members of the signalproducing system or some of the members of the signal producing system,for example, a second enzyme, can be present on the second bibulousstrip. See, for example, and U.S. patent application Ser. No. 733,013filed May 10, 1985 now U.S. Pat. No. 4,837,395 (Jun. 6, 1989), thedisclosures of which are incorporated herein by reference in theirentirety.

In another embodiment of the present invention a first bibulous striphaving an analyte analog bound to it is contacted at a contact portionwith an aqueous medium containing a sample suspected of containing ananalyte and an antibody for the analyte. The first bibulous strip is inliquid receiving relationship with an absorbent member, and the mediumis allowed to traverse the first bibulous strip and a portion of theabsorbent member. The liquid receiving relationship between theabsorbent member and the first bibulous strip is terminated and thefirst bibulous strip is brought into liquid receiving relationship witha second bibulous strip. The contact portion of the first bibulous stripis brought into contact with a second aqueous medium containing anantibody-enzyme conjugate which binds to the antibody for the analyte.The second bibulous strip contains a receptor for an antibody for theanalyte, for example, protein A.

If analyte is present in the sample, the analyte binds to itscomplementary antibody and, consequently, this complex does not becomebound to the first bibulous strip. However, if analyte is not present inthe sample or is present at or below a predetermined detectible amount,then antibody for the analyte binds to the analyte analog on the firstbibulous member. When the first bibulous strip is contacted with thesecond aqueous medium containing the antibody-enzyme conjugate, theconjugate becomes bound to the first bibulous strip if antibody foranalyte is bound. Depending upon the amount of antibody for analytebound to the first bibulous member, which in turn is related to theamount of analyte present in the sample, an amount of the conjugatemigrates to the second bibulous strip where it is captured by thereceptor for antibody.

After the medium has traversed the first bibulous strip and the secondbibulous strip, the second bibulous strip is treated as described abovein the previous embodiment in order to determine enzyme activity on thesecond bibulous strip. Again, the distance of migration of the conjugateon the second bibulous strip is related to the amount of analyte in thesample.

In another embodiment, a first bibulous strip with a portion havingantibody for analyte bound thereto is contacted with an aqueous mediumcontaining the sample of interest. The sample is allowed to traverse thefirst bibulous member being assisted by capillary migration into anabsorbent member in liquid receiving relationship with the firstbibulous member. After the traversal has taken place, the liquidreceiving relationship between the first bibulous member and theabsorbent member is terminated, and the first bibulous member is broughtinto liquid receiving relationship with a second bibulous member havingantibody for analyte bound thereto. The first bibulous member is thencontacted with an aqueous medium containing enzyme labeled analyteanalog. This second aqueous medium traverses the first bibulous memberand the second bibulous member.

If analyte is present in the sample, the analyte binds to the firstbibulous member in the first traversal and the antibody binding sites onthe first bibulous member become occupied. Thus, when the second aqueousmedium traverses the first bibulous member, the enzyme labeled analyteanalog does not bind to the first bibulous member but rather migrates tothe second bibulous member in direct relationship to the amount ofanalyte present in the sample. Again, the second bibulous member istreated to detect enzyme activity and to permit determination of thedistance of migration, which is then related to the amount of analyte inthe sample.

The present invention has application to a wide diversity of protocolsfor determining the presence and/or amount of one or more analytes in asample. One example in accordance with the present invention is an sbpmember in a first aqueous medium that is caused to become bound to afirst bibulous member by contacting a portion of the bibulous memberwith the medium. The first bibulous member is in liquid receivingrelationship with an absorbent member. The medium traverses the firstbibulous member and at least a portion of the absorbent member bycapillary action. Next, the first bibulous member is caused to come intoliquid receiving relationship with a second bibulous member. A componentin a second aqueous medium is caused to become non-diffusively bound toa second bibulous member in relation to the presence of analyte in thefirst medium. A portion of the first bibulous member is contacted withthe second aqueous medium under conditions wherein the second mediumtraverses the first bibulous member by capillary action and at least aportion of the second bibulous member. The presence of the component onat least a portion of, or the distance the component has migrated on,the second bibulous member is then determined to determine the presenceand/or amount of analyte in the sample. Protocols such as thosedescribed in U.S. patent application Ser. Nos. 904,597, filed Sep. 5,1986, now U.S. Pat. No. 4,959,307 (Sep. 25, 1990); Ser. No. 928,233,filed Nov. 7, 1986, now U.S. Pat. No. 5,030,558 (Jul. 9, 1991); thedisclosures of which are incorporated herein by reference in theirentirety, can be modified in accordance with the present invention todetermine one or more analytes in a sample.

In carrying out an assay for determining the amount of component, forexample, a metal ion, or an analyte in a liquid, usually aqueous,medium, the protocol will generally involve forming a combination in aliquid medium of the component and a first bibulous member zone ("firstzone"). The component of interest may be found in a wide variety ofsources such as chemical processing streams, media containingpollutants, waste water, physiologic fluids, and so forth. The firstzone contains a predetermined level of a reagent interreactive with thecomponent, for example, a chelator for the metal ion, or an sbp memberfor an analyte, non-diffusively bound thereto. In one approach a contactportion of the first zone is contacted with the liquid medium. Wettingof the first zone by capillary action is allowed to continue until themedium and the component have traversed all of the first zone andmigrated into a second bibulous member zone ("second zone"). The secondzone can be part of the same bibulous member as said first zoneprovided, however, that the composition of reagents bound to the twozones are different. Alternatively, the second zone can be part of aseparate bibulous member that is brought into contact with the firstzone to allow the medium and the component to migrate into the secondzone by capillary migration.

Next, the second zone is examined to determine the distances ofmigration of the medium and the component. Generally, this may beaccomplished by contacting the zone with a detection agent unless thecomponent is otherwise directly detectible or the detection agent isbound to the zone. The detection agent can be a developer solution suchas that for providing an enzyme substrate where the component containsan enzyme such as in the case of an enzyme labeled ligand analog. Wherethe component is an inorganic molecule, the detection agent can be asubstance or substance that interreacts with the component to produce asignal such as a color. For example, oxidation--reduction reactions canbe employed having as one member thereof a component where the reactionresults in the generation of a colored product.

The detection agent will frequently be present in an aqueous mediumwhich is contacted with the second zone by, for example, dipping thesecond zone into the medium containing the detection agent, applying themedium containing the detection agent to the second zone such as byspraying and the like, and so forth.

Sufficient time is provided so that the appropriate interaction can takeplace to the extent necessary. The time will depend on the nature of theinteraction such as chemical or physical, the interactivity of themolecules involved, and the like.

After the appropriate level of signal has developed, the distances ofmigration of the component into the second zone or the difference in thedistances of migration of the medium and the component into the secondzone can be measured. This distance or difference is related to theamount of the component in the liquid medium or the amount of reagent.For example, standard curves can be prepared utilizing known amounts ofcomponents and reagents and unknown amounts can be obtained by acomparison of the distance or difference referred to above with thestandard curve.

In a qualitative assay following the traversal of the first and secondbibulous members by the second medium, the distance the component hastraversed the second bibulous member is determined and is related to theamount of analyte in the sample. Where the component contains a label,the distance traversed can be determined from the presence of adetectible signal in the traversed area. If the label is, for example, afluorophore, the signal can be detected directly at the second bibulousmember. Where detection agents form part of the signal producing meansthat includes the label, the detection agents may be bound to the secondbibulous member or the member can be contacted with a solution of thedeveloper agent.

In a quantitative assay, generally, a sufficient time is allowed toelapse prior to measuring the signal to produce an amount of the signalproducing compound required to define the distance along the secondbibulous member over which the component has traversed. Once thedetectible signal has been produced, the amount of analyte in the sampleor the amount of reagent or component can be determined. Thus, thedistance between the furthest point of traversal of the medium and thefurthest point of traversal of the component on the chromatograph is ameasure of the amount of analyte in the sample, or of component, or ofreagent. The region of the second bibulous member traversed by thecomponent will usually be observed as a substantially uniform detectiblesignal throughout the region in which the component is present.

The present invention also includes devices for analyzing for thepresence or amount of an analyte or a reagent or component of interest.Referring to FIG. 1, the devices comprise a first bibulous member (10),an absorbent member (18), and a second bibulous member (20). The firstbibulous member is capable of being an alternating liquid receivingrelationship with the absorbent member and the second bibulous member.In an alternate embodiment, the present devices further comprise a meansfor alternating the liquid receiving relationship. As mentioned above,this means can be physical or mechanical and may be automated ornon-automated. In one embodiment of a device in accordance with thepresent invention, the second bibulous member is an immunochromatograph.

As a matter of convenience, the reagents and devices employed in thepresent invention can be provided in a kit in packaged combination inpredetermined amounts for use in conducting the present method inassaying for an analyte in the sample. For example, a kit useful in thepresent method can comprise in packaged combination a device asdescribed above and a component or a component and a first reagent, asdescribed above. Where an enzyme is used as a label, the reagents caninclude enzyme labelled analyte analog and a developer solution, whichcan contain substrate for the enzyme or precursors therefor includingany additional substrates, enzymes and cofactors and any reactionpartner of the enzymic product required to provide the detectiblechromophore or fluorophore. In addition, other additives, such asancillary reagents, may be included, for example, stabilizers, buffers,and the like. The relative amounts of the various reagents may be variedwidely to provide for concentrations in solution of the reagents whichsubstantially optimize the sensitivity of the assay. One or more of thereagents can be provided as dry powders, usually lyophilized, includingexcipients, which on dissolution will provide for a reagent solutionhaving the appropriate concentrations for performing the assay. Eachreagent can be packaged in separate containers or some reagents can becombined in one container where reactivity and shelf life will permit.

EXAMPLES

The invention is demonstrated further by the following illustrativeexamples. Parts and percentages are by weight to volume unless indicatedotherwise.

EXAMPLE I

PVA--polyvinyl alcohol

MES buffer--2-morpholinoethane sulfonic acid

TNBS--trinitrobenzenesulfonic acid

HEPES buffer--hydroxyethylpiperazine ethanesulfonic acid

DMF--dimethylformamide

PBS--phosphate buffered saline-10 mM sodium phosphate, 150 mM sodiumchloride, pH 7

NHS--N-hydroxysuccinimide

Ig--immunoglobulins

IgG--immunoglobulin G

MATERIALS

Nitrocellulose membrane (12μ pore size) was obtained from Schleicher andSchuell. Affinity purified rabbit anti-Mouse Ig was obtained from Zymed.Glutaraldehyde (25% aqueous) was obtained from Sigma. Bovine IgG wasobtained from Miles Diagnostics. Monoclonal anti-fluorescein 11H7 andmonoclonal anti-digoxin 2H6 were prepared according to standard methods[Galfre, et al., Nature (1977) 266:550], using fluorescein-labeledbovine IgG and digoxin-labeled keyhole limpet hemocyanin, respectively,as immunogens. Phosphate buffer was 0.1M sodium phosphate, 0.2M sodiumchloride, pH 7.3 Sodium sulfate buffer was phosphate buffer whichadditionally contained 9% w/w sodium sulfate. Horseradish peroxidase(HRP) was obtained from Shinko American. Blotting paper was obtainedfrom Bio-Rad (Cat. No. 1650921). Carboxyfluorescein was from Kodak.

Preparation of Immobilized Anti-Fluorescein

Nitrocellulose membrane (7.5×9 cm) was incubated with 50 mL of asolution of 40 μg/mL rabbit anti-mouse Ig and 1 mg/mL bovine IgG insodium sulfate buffer for 30 min. The membrane was washed with 50 mL ofsodium sulfate buffer for 15 min., then fixed by incubating with 50 mLof 62 μg/mL glutaraldehyde in sodium sulfate buffer for 2 hr. Themembrane was washed twice, for 15 min each, with 50 mL phosphate buffer,then incubated with 50 mL of 100 μg/mL monoclonal anti-fluorescein 11H7and 1 mg/mL bovine IgG in phosphate buffer for 1 hr. The membrane waswashed with 50 mL phosphate buffer for 15 min, washed with 50 mLdeionized water for 15 min, and incubated with 50 mL of 0.2 mg/mL 20/30PVA. The membrane was then dried at 50° C. for 15 min.

Preparation of Immobilized Anti-Digoxin

Nitrocellulose membrane (27.5×9 cm) was laminated onto 3-mil vinylacetate film using 3M 9460 transfer adhesive and then incubated with 250mL of a solution of 1 μg/mL rabbit anti-mouse Ig and 1 mg/mL bovine IgGin sodium sulfate buffer for 30 min. The membrane was washed with 250 mLof sodium sulfate buffer for 15 min, then fixed by incubating with 250mL of 62 μg/mL glutaraldehyde in sodium sulfate buffer for 2 hr. Themembrane was washed twice, for 15 min each, with 250 mL phosphatebuffer, then incubated with 250 mL of 1 μg/mL monoclonal anti-digoxin2H6 and 1 mg/mL bovine IgG in phosphate buffer for 1 hr. The membranewas washed with 250 mL phosphate buffer for 15 min, washed with 250 mLdeionized water for 15 min, and incubated with 250 mL of 0.2 mg/mL 20/30PVA. The membrane was then dried at 50° C. for 15 min.

Preparation of Blocked Nitrocellulose Membrane

Nitrocellulose membrane (7.5×9 cm) was incubated with 50 mL of asolution of 1 mg/mL bovine IgG in sodium sulfate buffer for 30 min. Themembrane was washed with 50 mL of sodium sulfate buffer for 15 min, thenfixed by incubating with 50 mL of 62 μg/mL glutaraldehyde in sodiumsulfate buffer for 2 hr. The membrane was washed twice, for 15 min each,with 50 mL phosphate buffer, then incubated with 50 mL of 1 mg/mL bovineIgG in phosphate buffer for 1 hr. The membrane was washed with 50 mLphosphate buffer for 15 min, washed with 50 mL deionized water for 15min, and incubated with 50 mL of 0.2 mg/mL 20/30 PVA. The membrane wasthen dried at 50° C. for 15 min.

Preparation of Digoxin-labeled HRP

Ethylenediamine (3M, pH 5.0; 3.33 mL), and1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (1M, 0.1 mL) were added toa solution of HRP (50 mg in 6.6 mL of 138 mM MES buffer, pH 5.0), andthe mixture was stored at room temperature for 15 hr. The resultingaminated HRP was purified by chromatography on Sephadex G-25 using 0.02%aqueous sodium azide as eluant, and was found to have an average of 11amino groups per HRP (TNBS titration). Aminated HRP (37.9 mg) in HEPESbuffer(200 mM, pH 8.0; 7 mL) was treated with three 95 μL-portions ofsuccinic anhydride (2M in DMF) added at 10 min intervals, and 5 minafter each addition a 190-μL portion of 1M sodium hydroxide was added tomaintain the pH at 8. The succinylated aminated HRP was purified bychromatography on Sephadex G-25 using 0.02% aqueous sodium azide aseluant. The succinylated aminated HRP was reaminated by treating thematerial (30.8 mg) in MES buffer (70 mM, pH 5.0; 4.1 mL) withethylenediamine (3M, pH 5.0; 2.07 mL) and1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (1M, 0.062 mL) for 6 hr atroom temperature. The reaminated HRP was purified by chromatography onSephadex G-25 using 0.02% aqueous sodium azide as eluant and was foundto have 15.5 amino groups per HRP (TNBS titration). The reaminated HRP(2 mg/mL in PBS, pH 7.0) was reacted with a 200-fold excess of the NHSester of 3-digoxigenin-O-carboxymethyloxime and then exhaustivelydialyzed. The resulting digoxin-labeled HRP had 12 digoxins/HRP (basedon titration of residual amino groups with TNBS).

Preparation of Fluorescein-labeled Anti-Digoxin 2H6

Monoclonal anti-digoxin 2H6 (7.3 mg/mL, 0.2 mL) in phosphate buffer wastreated with the NHS ester of 6-carboxyfluorescein (20 mM in DMF; 5 μL)and incubated for 5 min at room temperature, then 16 hr at 4° C. thefluorescein-labeled anti-digoxin was purified by chromotography onSephadex G-25 using phosphate buffer as eluant, and was found to have3.6 fluoresceins per antibody.

Device, (Refer to FIG. 1)

First bibulous member (10)--an end portion, consisting of a 6 mm×6 mmpiece of blocked nitrocellulose membrane (12), and a capture pad,consisting of a 6 mm×10 mm piece of immobilized anti-fluorescein (14),were laminated onto 3-mil acetate film (16) such that the two sectionsoverlapped by 1 mm.

Absorbent member (18)--a 12 mm×50 mm strip of blotting paper.

Second bibulous member (20) (quantitating strip)--a 6 mm×90 mm strip ofimmobilized anti-digoxin.

During the assay the first bibulous member was first in liquid receivingrelationship (overlap by 1 mm) with the absorbent member (referred tobelow as the sample uptake step) and then in liquid receivingrelationship with the second bibulous member (referred to below as thequantitation step).

Assay Protocol

The assay was carried out in two steps--a sample uptake step and aquantitation step. For the sample uptake step, sample (100 μL) andfluorescein-labeled anti-digoxin 2H6 (1.25 μg/mL, 50 μL) were dispensedinto the bottom of a 16×100 mm test tube and mixed. An end portion ofthe first bibulous member of an assay device such as that depicted inFIG. 1 was placed in the test tube and the liquid was allowed to wickalong the first bibulous member and into the absorbent. After all of theliquid had been taken up by the device, the contact between absorbentand the first bibulous member was terminated. For the quantitation step,the quantitating strip was placed in contact with the upper edge of thecapture pad. The end portion of the first bibulous member of the assaydevice was then placed in a test tube containing digoxin-labeled HRP(250 ng/mL, 100 μL) and liquid was allowed to pass through the capturepad into the quantitating strip until the quantitating strip was filledwith liquid. The quantitating strip was then removed from the device andplaced in a developer solution (containing 0.5 mg/mL dicarboxidine, 10mM hydrogen peroxide in 100 mM sodium citrate, pH 5.0) until a bluecolor had developed on a portion of the quantitating strip. The length(migration height) of the blue area was measured and was related to theconcentration of digoxin in the sample.

Assay Results

A series of assays was performed using serum samples containing knownconcentrations of digoxin (Column Digoxin calibrators, Syva Company).The results are summarized in Table 1.

                  TABLE 1                                                         ______________________________________                                        Digoxin Concentration                                                                          Migration Height                                             (ng/mL)          (mm)                                                         ______________________________________                                        0                20                                                           0.5              24                                                           1                29                                                           2                39                                                           3                48                                                           4                55                                                           ______________________________________                                    

The above results indicate that an accurate and sensitive assay fordigoxin can be conducted utilizing the method and devices of the presentinvention. Increasing concentrations of digoxin resulted in greatermigration height observed on the quantitating strip.

EXAMPLE 2

Measurement of Concentration of Digoxin-Labeled HRP

Using devices as described above in Example 1, 100 μl of a 400 ng/mlsolution of fluorescein-labeled anti-digoxin was passed by capillaryaction through the first bibulous member into the absorbent. Solutionsof digoxin-labeled HRP of varying concentrations were analyzed with thisdevice by passing them by capillary action through the first bibulousmember into the second bibulous member. The migration distances were afunction of the concentration of digoxin-labeled HRP as shown in thefollowing table (Table 2).

                  TABLE 2                                                         ______________________________________                                        Concentration of Digoxin-Labeled HRP                                                               Migration Distance                                       (ng/mL)              (mm)                                                     ______________________________________                                        100                   0                                                       150                   7                                                       200                  14                                                       250                  18                                                       300                  23                                                       ______________________________________                                    

EXAMPLE 3

Measurement of Concentration of Fluorescein-Labeled Anti-Digoxin

Using devices as described above in Example 1, 120 μl of solutions offluorescein-labeled anti-digoxin of varying concentrations were passedby capillary action through the first bibulous member into theabsorbent. The analyses were completed by passing solutions of 275 ng/mldigoxin-labeled HRP through the first bibulous member into the secondbibulous member by capillary action. The migration distances were afunction of the fluorescein-labeled anti-digoxin concentrations as shownin the following table (Table 3).

                  TABLE 3                                                         ______________________________________                                        Concentration of Fluorescein-Labeled                                                                Migration Distance                                      Anti-Digoxin (ng/mL)  (mm)                                                    ______________________________________                                         660                  35                                                      1000                  20                                                      ______________________________________                                    

EXAMPLE 4

Measurement of Magnesium Chloride Concentration

Devices are prepared in accordance with FIG. 1 where the second bibulousmembers are pieces (0.5×10 cm) of untreated nitrocellulose membrane (12μpore size). The first bibulous member (0.5×1.0 cm) are prepared bytreating Whatman 1C chromatography paper sequentially with carbonyldiimidazole (R. F. Zuk, V. K. Ginsberg, T. Houts, et al. (1986) ClinChem, 31:1144-1150), ethylenediamine, and the anhydride ofdiethylenetriaminepentaacetic acid in methylene chloride. The membersare dried, washed repeatedly with water, and redried.

Using these devices, aqueous solutions of varying magnesium chlorideconcentration are passed through the first bibulous member into thesecond bibulous member by capillary action. Magnesium ion is taken up bythe chelating group on the first bibulous member. The magnesiumchloride-containing zones on the second bibulous member are visualizedby spraying the strips with a dilute solution of Eriochrome Black Tbuffered at pH 9. The migration distances are a function of themagnesium chloride concentration.

EXAMPLE 5

Measurement of Sodium Iodate Concentration

The first bibulous member of devices similar to those of the precedingexample are treated with excess aqueous ferrous chloride and then washedand dried. Aqueous solutions of sodium iodate of varying concentrationsare passed through the first bibulous member by capillary action intothe second bibulous member. Sodium iodate is reduced to sodium iodide bythe chelated ferrous ion. After the second bibulous member has filledwith liquid, the unreduced sodium iodate-containing zones are visualizedby contacting the second bibulous member with a developer solutioncontaining starch and sodium iodide. The migration distances are afunction of the sodium iodate concentration.

EXAMPLE 6

Measurement of Ferrous Chloride Concentration

Using devices similar to those described in Example 4, 50 μl of aqueousferrous chloride solutions of varying concentrations are passed throughthe first bibulous member by capillary action into the absorbent strips.Solutions of 0.10 mM sodium iodate are then passed through the firstbibulous member by capillary action into the second bibulous member. Theunreduced sodium iodate-containing zone on the second bibulous member isvisualized as in the previous example. The migration distances are afunction of the ferrous chloride concentration.

EXAMPLE 7

Alternate Method for Measurement of Ferrous Chloride Concentration

The procedure described above in Example 6 for the measurement offerrous chloride concentration is followed, except that 5 μl samples ofthe aqueous ferrous chloride solution are applied directly to the firstbibulous member rather than passing them through the first bibulousmember. The migration distances are a function of the ferrous chlorideconcentration.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. A device for conducting an assay, which devicecomprises:a first bibulous member strip, an absorbent member strip, asecond bibulous member strip, a portion of said first bibulous memberstrip being in alternating liquid receiving relationship, by directcontact, with a portion of said absorbent member strip and with aportion of said second bibulous member strip.
 2. The device of claim 1,which further comprises means for alternating said liquid receivingrelationship, said means being adapted for terminating a liquidreceiving relationship between a portion of said first bibulous memberstrip and a portion of said absorbent member strip and creating a liquidreceiving relationship between a portion of said bibulous member stripand a portion of said second bibulous member strip.
 3. The device ofclaim 1 wherein said second bibulous member is an immunochromatograph.4. A kit for conducting an assay, which comprises in packagedcombination:(a) the device of claim 1 and (b) reagents for conductingsaid assay.